Friday, 1 May 2020

CRISPR gene cuts may offer new way to chart human genome

In
search of new ways to sequence human genomes and read critical alterations in
DNA, researchers at Johns Hopkins Medicine say they have successfully used the
gene cutting tool CRISPR to make cuts in DNA around lengthy tumor genes, which
can be used to collect sequence information.

The
researchers say that pairing CRISPR with tools that sequence the DNA components
of human cancer tissue is a technique that could, one day, enable fast,
relatively cheap sequencing of patients' tumors, streamlining the selection and
use of treatments that target highly specific and personal genetic alterations.

In
conventional genome sequencing, scientists have to make many copies of the DNA
at issue, randomly break the DNA into segments, and feed the broken segments
through a computerized machine that reads the string of chemical compounds
called nucleic acids, made up of the four "bases" that form DNA, and
are lettered A, C, G and T. Then, scientists look for overlapping regions of
the broken segments and fit them together like tiles on a roof to form long
regions of DNA that make up a gene.

In
their experiments, Timp and M.D./Ph.D. student Timothy Gilpatrick were able to
skip the DNA-copying part of conventional sequencing by using CRISPR to make
targeted cuts in DNA isolated from a sliver of tissue taken from a patient's
breast cancer tumor.

Then,
the scientists glued so-called "sequencing adaptors" to the
CRISPR-snipped ends of the DNA sections. The adaptors serve as a kind of handle
that guide DNA to tiny holes or "nanopores" which read the sequence.

By passing DNA
through the narrow hole, a sequencer can build a read-out of DNA letters
based on the unique electrical current that occurs when each chemical code
"letter" slides through the hole.

Among
10 breast cancer genes the team focused on, the Johns Hopkins scientists were
able to use nanopore sequencing on breast cancer cell lines and tissue samples
to detect a type of DNA alteration called methylation, where chemicals called
methyl groups are added to DNA around genes that affect how genes are read.

The
researchers found a location of decreased DNA methylation in a gene called
keratin 19 (KRT19), which is important in cell structure and scaffolding.
Previous studies have shown that a decrease in DNA methylation in KRT19 is
associated with tumor spread.

In
the breast cancer cell lines they studied, the Johns Hopkins team was able to
generate an average of 400 "reads" per basepair, a reading
"depth" hundreds of times better than some conventional sequencing
tools.

Among
their samples of human breast cancer tumor tissue taken at biopsies, the team
was able to produce an average of 100 reads per region. "This is certainly
less than what we can do with cell lines, but we have to be more gentle with
DNA from human tissue samples because it's been frozen and thawed several
times," says Timp.

In
addition to their studies of DNA methylation and small mutations, Timp and
Gilpatrick sequenced the gene commonly associated with breast cancer: BRCA1,
which spans a region on the genome more than 80,000 bases long. "This gene
is really long, and we were able to collect sequencing reads which went all the
way through this large and complex region," says Gilpatrick.

"Because
we can use this technique to sequence really long genes, we may be able to
catch big missing blocks of DNA we wouldn't be able to find with more
conventional sequencing tools," says Timp.

In
addition to its potential to guide treatment for patients, Timp says the
combination of CRISPR technology and nanopore sequencing provides such depth
that it may help scientists find new disease-linked gene alterations specific
to one allele (inherited from one parent) and not another.

Timp
and Gilpatrick plan to continue refining the CRISPR/nanopore sequencing
technique and testing its capabilities in other tumor types.